Blood coagulation is a complex process including the sequential interaction of a series of components, in particular of fibrinogen, Factor II, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII and von Willebrand's factor. The loss of one of these components or the inhibition of its functionality may cause either an increased tendency of blood coagulation or an inability to clot, which may be life-threatening in some patients.
Factor VIII is a protein found in blood plasma which acts as a cofactor in the cascade of reactions leading to blood coagulation. A deficiency in the amount of Factor VIII activity in the blood results in the clotting disorder hemophilia A, an inherited condition primarily affecting males. Hemophilia A is currently treated with therapeutic preparations of Factor VIII derived from human plasma or manufactured using recombinant DNA technology. Such preparations are administered either in response to a bleeding episode or at frequent, regular intervals to prevent uncontrolled bleeding (prophylaxis).
Von Willebrand factor (vWF) circulates in plasma complexed with factor VIII. vWF complexed with Factor VIII stabilizes the Factor VIII protein and protects it from proteolytic degradation. Due to its function in platelet aggregation, vWF also directly interferes in blood coagulation. Von Willebrand deficiency (vWD) (also known as von Willebrand syndrome) results from either a deficiency or overexpression of vWF. Deficiency of vWF results in a disease similar to hemophilia due to the rapid degradation of Factor VIII lacking vWF cofactor.
Conventional methods for a therapy of Hemophilia A and von Willebrand syndrome use Factor VIII or vWF recovered from plasma or produced by recombinant sources, and there are a number of attempts to treat patients with purified Factor VII, vWF or factor VIII/vWF-complex. The development of antibodies against the administered exogenous protein can decrease the efficacy of treatment and presents a challenge to treatment of these patients. For example, anti-FVIII antibodies are especially prevalent in patients with severe and moderately severe hemophilia, which develop anti-FVIII antibodies at a frequency of 50% (Gilles et al., Blood 82:2452-61, 1993; Lacroix-Desmazes et al., J. Immunol. 177:1355-63, 2006).
Transgenic animal technology presents a unique opportunity to study the characteristics of human proteins in non-human animals. Recombinant DNA and genetic engineering techniques have made it possible to introduce and express a desired sequence or gene in a recipient animal making it possible to study the effects of a particular molecule in vivo and study agents that bind to the molecule. One procedure for producing transgenic mice requires the recovery of fertilized eggs from newly mated female mice and microinjection of the DNA of the gene of interest into the male pronucleus of the egg. The microinjected eggs are then implanted in the oviducts of one-day pseudopregnant foster mothers and allowed to proceed to term. See, for example, Wagner et al. P.N.A.S. U.S.A. 78:6376-6380 (1981), U.S. Pat. Nos. 4,873,191, and 7,294,755. Another procedure uses embryonic stem cells that are transfected with the gene of interest. Transfected embryonic stem cells are then injected into mouse blastocysts where they take part in the formation of all tissues, including the germ line, thus generating transgenic offspring. This approach, in combination with the homologous recombination technology, offers the possibility of altering embryonic stem cells in a controlled manner and therefore, of generating transgenic mice with a predetermined genome. See, for example: Baribault and Kemler. Embryonic stem cell culture and gene targeting in transgenic mice. Mol Biol Med. 6:481-92, 1989; Ledermann B. Embryonic stem cells and gene targeting. Exp Physiol. 85:603-13, 2000; Moreadith and Radford. Gene targeting in embryonic stem cells: the new physiology and metabolism. J Mol Med. 75:208-16, 1997.
Transgenic mice may be generated to express or overexpress a protein of interest (knock-in mice) or may be generated to delete a gene of interest (knock-out mice). Transgenic mice which express a human protein molecule allow for study of the human molecules in vivo. For example, Shi et al. (J Clin Invest. 116:1974-82, 2006) describe transgenic mice expressing a modified human FVIII protein (lacking the B-domain) designed to circumvent the problem of FVIII inhibitory antibodies which inhibit the activity of recombinant FVIII.
The advent of transgenic technology also allows for development of screening methods which would not be possible without the transgenic animals. For example, in order to study the development of antibodies to an exogenous protein, it is useful to have a model in which the subject is naturally tolerant to the molecule of interest. U.S. Pat. No. 5,470,560 describes a method for screening for the immunogenicity of a polypeptide using a transgenic mouse expressing the protein of interest and tolerant to the protein, administering the exogenous protein to the animal, and screening for antibodies specific for the polypeptide. International Patent Application No. WO2006/056769 describes a method for testing the immunogenicity of a mammalian antigen in an animal transgenic for the cognate MHC class II molecules which would naturally present the mammalian antigen.
Development of antibodies to protein therapeutics is a persistent problem when biopharmaceuticals are used for treatment of disorders. These antibodies often inhibit the activity of the protein therapeutic thereby reducing the efficacy of the treatment or requiring increasing doses of drug to maintain therapeutic levels. Because blood disorders such as hemophilia are often lifelong conditions, the appearance of antibodies specific for therapeutic blood clotting factors is particularly trying for the patients receiving the treatment and challenging for doctors treating these patients.
Thus, there exists a need in the art to develop methods to study the activity of human blood coagulation factors in vivo without study on human patients. Further, there remains a need in the art to determine if administration of exogenous therapeutic protein to a patient will result in production of antigen-specific antibodies which inhibit protein activity in vivo.